Background: ␣-Synuclein (␣-syn) is a major component of Lewy bodies, a pathologic marker of Parkinson's disease (PD) in post-mortem studies. The use of ␣-syn as a practical PD biomarker has been investigated by numerous researchers. However, reports of differences in ␣-syn levels in biofluids, such as cerebrospinal fluid, plasma, and saliva, between PD patients and controls are inconsistent. Recently, the measurement of ␣-syn oligomer levels has emerged as a novel approach to diagnose PD. Objective: Lysates and culture media from two different types of dopaminergic neuronal cells or urine samples from 11 non-PD and 21 PD patients were collected and analyzed. Methods: We developed and performed an enzyme-linked immuno-absorbent assay (ELISA) to detect various oligomeric ␣-syn using distinct pairs of antibodies. Results: We validated our ELISA using rotenone-induced alterations of ␣-syn levels in human dopaminergic neurons. Total urinary ␣-syn levels, measured using our ELISA method, showed no difference between PD and non-PD individuals, but a higher level of ␣-syn oligomer recognized by MJFR-14-6-5-2 in PD urine samples was observed. Levels of distinct oligomeric ␣-syn detected by ASyO5 were lower in PD urine samples. Three different ␣-syn ELISA results were analyzed with respect to the severity of PD, but only the correlation between total ␣-syn levels and PD index was significant.
Although brain organoids are an innovative technique for studying human brain development and disease by replicating the structural and functional properties of the developing human brain, some limitations such as heterogeneity and long-term differentiation (over 2 months) impede their application in disease modeling and drug discovery. In this study, we established simplified brain organoids (simBOs), composed of mature neurons and astroglial cells from expandable hPSC-derived primitive neural stem cells (pNSCs). simBOs can be rapidly generated in 2 weeks and have more homogeneous properties. Transcriptome analysis revealed that three-dimensional (3D) environment of simBOs facilitates the conversion of pNSCs to mature neuronal systems compared to a two-dimensional environment in the context of neurotransmitter release, synaptic vesicle formation, ion channels, calcium signaling, axonal guidance, extracellular matrix organization, and cell cycle. This result was correlated with the translocation of YAP1 into the cytoplasm by sensing matrix stiffness on the 3D models. Furthermore, we demonstrated that simBOs could easily be specified into midbrainlike simBOs by treatment with Shh and FGF8. Midbrain-like simBOs from a Parkinson's disease patient (LRRK2 G2019S)-derived pNSCs and gene-corrected (LRRK2 WT) control pNSCs represented disease-associated phenotypes in terms of increased LRRK2 activity, decreased dopaminergic neurons, and increased autophagy. Treatment with the LRRK2 inhibitor, PFE-360, relieved the phenotype of Parkinson's disease in midbrainlike simBOs. Taken together, these approaches could be applied to large-scale disease models and alternative drug-testing platforms.
In Parkinson’s disease (PD) research, human neuroblastoma and immortalized neural cell lines have been widely used as in vitro models. The advancement in the field of reprogramming technology has provided tools for generating patient-specific induced pluripotent stem cells (hiPSCs) as well as human induced neuronal progenitor cells (hiNPCs). These cells have revolutionized the field of disease modeling, especially in neural diseases. Although the direct reprogramming to hiNPCs has several advantages over differentiation after hiPSC reprogramming, such as the time required and the simple procedure, relatively few studies have utilized hiNPCs. Here, we optimized the protocol for hiNPC reprogramming using pluripotency factors and Sendai virus. In addition, we generated hiNPCs of two healthy donors, a sporadic PD patient, and a familial patient with the LRRK2 G2019S mutation (L2GS). The four hiNPC cell lines are highly proliferative, expressed NPC markers, maintained the normal karyotype, and have the differentiation potential of dopaminergic neurons. Importantly, the patient hiNPCs show different apoptotic marker expression. Thus, these hiNPCs, in addition to hiPSCs, are a favorable option to study PD pathology.
Reprogramming with episomal vectors is an easy, safe, and cost-effective method to generate exogenous DNA-free (exogene-free) induced pluripotent stem cells (iPSCs). However, the genomic integration of exogenes is observed occasionally. Additionally, the removal of episomal DNA takes more than 70 days in established iPSCs. Here, we inserted the cytosine deaminase (CD) gene from yeast into episomal vectors and used them to reprogram human fibroblasts into iPSCs. These new episomal vectors (CD episomal vectors) were eliminated from the generated iPSCs as early as seven days after 5-fluorocytosine (5-FC) treatment. We also found that cells with the integration of the CD gene perished within two days of 5-FC treatment. In addition, we generated exogene-free induced neural stem cells after one passage by direct reprogramming with CD episomal vectors combined with 5-FC treatment. Conclusively, our novel method allows the rapid and easy isolation of exogene-free reprogrammed cells and can be applied to disease modeling and clinical applications.
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